Identification of valid targets for intervention in the treatment of diabetes requires knowledge of the mechanisms of metabolic signal generation leading to stimulation of insulin secretion. Changes in Ca2+ alone cannot explain the wide dynamic range of glucose stimulated insulin secretion. We find no difference in glucose-induced Ca2+ transient between 8, 10 and 12 mM glucose where secretion continues to increase. In addition, the mechanism by which increases in glucose from 5 to 7 mM stimulate more secretion in the presence of incretins without a further change in Ca2+ is not established. Moreover, neither changes in ATP, membrane potential (DY) nor Ca2+ have been shown to explain the potent ability of the mitochondrial agonist methyl succinate (MeS) to induce secretion. We hypothesize that NADPH and O2-, in the low physiological range, are common metabolically generated signals that regulate a cytosolic redox network to control protein function. This network could play a role in such still unexplained processes as vesicular docking, Ca2+ handling or plasma membrane (PM) repolarization. We find rapid concentration-dependent generation of superoxide (02-) in isolated islets and islet cells in response to MeS, glucose and glucose plus incretins. Both MeS and glucose increase [unreadable]-cell cytosolic redox state via increases in NADPH and the glutathione (GSH) and thioredoxin (TRX) network that are necessary for the handling of 02- following its conversion to H202 by superoxide dismutase. A role for the NADPH and O2--regulated TRX network in insulin secretion is also indicated by altered secretory responsiveness in mice with a defect in this system. The goal of the proposed work is to determine the importance of NADPH and 02- and the redox networks in physiological insulin secretion by pursuing the following aims: Aim 1. What are the signals that accompany secretion in response to small increments in glucose and in the presence of incretins? This aim will also evaluate the phasing of the responses. Aim 2. What are the mitochondrial and cytosolic metabolic signals that accompany MeS-induced insulin secretion? Aim 3. Redox mediators and their downstream targets in GSIS. We will determine whether physiological alterations in 02- or NADPH alter TRX or GSH activity or DYPM and whether separately altering TRX, GSH or voltage-sensitive K+- channel activity alters insulin secretion. [unreadable] [unreadable]